Antibodies to gangliosides have been implicated in many different autoimmune neuropathies. For example, serum IgM antibodies to GM ganglioside have been found in a patient with lower motor neuron syndrome (Adams, D. et al., J. Neurol. Neurosurg. Psychiatry 56:982-987 (1993)), and are common in multifocal motor neuropathy (Kornberg, A. J. and Pestronk, A., Ann. Neurol. 37:S43-S50 (1995); Komberg, A. J. and Pestronk, A., Muscle Nerve 17:100-104 (1994); Cetacea, S. A. et al., Neurology 40:1067-1072 (1990); Adams, D. et al., Neuroimmunology 32:223-230 (1991); Taylor, B. V. et al., Neurology 46:951-955 (1996)). Autoantibodies against GM- and GM-gangliosides have been found in conjunction with chronic idiopathic demyelinating polyneuropathy (CID) in conjunction with systemic lupus erythematosus (SLE) (Sindem, E. et al., Acta. Neurol. Scand. 83:399-402 (1991)). Anti-GQ1B ganglioside antibodies were identified in patients with Fisher""s syndrome (Yuri, N. et al., Neurology 43:414-417 (1993)). Sera from patients with Gillian-Barr. syndrome has been found to have antibodies to various gangliosides, including antibodies to GM1B ganglioside (see, e.g., Cathouse, K. et al., J. Neurol. Sci. 156:99-101 (1998)).
Enzyme-linked immunosorbent assays (ELISA) have been used for identification of antibodies to gangliosides (see, for example, identification of GM ganglioside in Pestronk, A. et al. Ann. Neurol. 27:316-326 (1990)); U.S. Pat. No. 5,443,952; Aenaeus-Kanaf, A. B. et al., Neurochem. Inv. 20(3):353-357 (1992)). However, high background values frequently interfere with accurate assessment of the amount of anti-ganglioside antibodies (Ravindranath, MH et al., J. Immunological Methods 169:257-272 (1994)). Reliable measurement of anti-ganglioside antibodies is critical for correct diagnosis of neuropathies, particularly motor neuropathies.
The present invention pertains to methods of determining, in a test sample, the amount of antibodies directed against a specific nervous system antigen or antigens, using a modified solid-phase reactant. The method utilizes a solid-phase reactant, such as a microtiter plate, that is modified with carbonyl groups attached to its surface. One or more gangliosides of interest (e.g., asialo GM ganglioside, GM1 ganglioside, GM2 ganglioside, GM3 ganglioside, GD1a ganglioside, GD1B ganglioside, GD2 ganglioside, GD3 ganglioside, GQ1b ganglioside, and/or GT1b ganglioside)are linked to the modified solid-phase reactant by an amide bond between an amino group of the ganglioside and a carbonyl group attached to the solid-phase reactant. One or more control antigens, such as other glycolipids, glycoproteins or carbohydrates, can also be attached on the surface of the modified solid-phase reactant. The modified solid-phase reactant having ganglioside(s) of interest linked thereon is contacted with a test sample, such as a test sample of a bodily fluid (e.g., blood, serum, cerebrospinal fluid, or urine) from an individual, under conditions such that any antibody to the ganglioside(s) of interest that may be present in the test sample can bind to the ganglioside(s) of interest linked to the modified solid-phase reactant. The amount of antibodies in the test sample to the ganglioside(s) of interest is then determined using standard methods, such as enzyme-linked immunosorbent assay (ELISA) or another appropriate solid-phase assay. If a control antigen is attached on the modified solid-phase reactant, the level of antibodies in the test sample to the control antigen, can also be determined using the same methods. Specific reactivity of antibodies to the ganglioside of interest is determined by the amount of antibody binding to the ganglioside of interest that is above the amount of antibody binding to the control antigen. The methods can be used for diagnosing a neuropathy in an individual. The amount of antibody to a ganglioside of interest in a test sample from the individual is determined using the methods. An amount of antibody to a ganglioside of interest that is greater, by an amount that is statistically significant, than the amount of antibody to the ganglioside of interest in a control sample, is indicative of the presence of the neuropathy. Alternatively, an amount of antibody to a ganglioside of interest that is equal to or greater than an established reference amount is indicative of the presence of the neuropathy.
The invention also pertains to test kits, containing modified solid-phase reactants, for use in the methods of the invention.
The high sensitivity and specificity of the methods can clarify the differential diagnosis of neuropathies and reduce the need for time-consuming and expensive electrophysiological evaluation. Furthermore, a modified solid-phase reactant having carbonyl groups attached to its surface allows the use of a smaller amount of ganglioside than the amount which would otherwise be necessary to perform similar assays with a solid-phase reactant not having this modification. In addition, a modified solid-phase reactant having carbonyl groups attached to its surface can be coated with a ganglioside of interest without a need for toxic solvents; the coating is not affected by humidity, and yields consistent and reproducible assay results.